User:Alexander Rudecki/Sandbox 1: Difference between revisions

[[Image:Cropped_dimer.jpg|thumb|400px|right|Figure 2. A 3D graphical representation displaying the homodimer glutaminyl cyclase from ''Drosophila melanogaster'' (PDB: 4F9U). Secondary structure is depicted by red (α-helix) and yellow (β-strand) ribbons, glycosyl groups are coloured pink, while hydrogen bonds between the two monomers are shown by dotted green lines. The active site of QC contains a chelated zinc ion represented by a gray sphere. Also bound to the active site of this crystal structure and depicted as blue is the inhibitor 1-(3,4-dimethoxyphenyl)-3-[3-(1H-imidazol-1-yl)propyl]thiourea.]][[Image:DromeQCActiveSite.png|thumb|300px|left|Figure 4. A comparison between the active sites of DromeQC crystalized either with a PBD150 inhibitor (right, PDB: 4F90) or without (left, PDB: 4FWU). Protein loops surrounding the active site are denoted in dark blue, providing a scaffold for a catalytic Zn²⁺ (gray) to be chelated by three residues (light blue).The PBD150 inhibitor (red) involve interactions with W296 (yellow), F292 (green), W176 (beige) and D271 (pink).]]

As shown above, DromeQC is made up of 2 identical, independent monomers that come together to form an asymmetric <scene name='58/580851/Two_chains/2'>homodimer</scene> (Figure 1). The subunits are connected via 4 hydrogen bonds (Chain A→Chain B: ARG35 NH2→GLU64 OE2, ARG43 NH2→ASN71 O, ARG43 NH2→PHE75 O, ARG43 NH1→PHE75 O) and surface complementarity (Figure 2). The subunits exhibit a globular α/β-hydrolase fold, characterized by a central twisted β-sheet motif consisting of 5 parallel strands (β1 and β3-β6) and an antiparallel β2 strand (Figures 2&3). This β-center is flanked by 9 surrounding α-helices; 2 fill the concave face (α5, α7), 7 fill the convex face (α1- α5, α8, α9) with one helix at the edge (α6) of each monomer. DromeQC is glycosylated (with up to 7 carbohydrate moieties) at the N42 position. These polysaccharide tags increase the solubility of DromeQC, and appear to have no affect of protein activity<ref name="main"/>.

Binding of PBD150 to DromeQC utilizes π-π, arene-H, and hydrogen bonding interactions (Figure 4). The dimethoxyphenyl phenyl group of PBD150 is stabilized by π-π interactions with F292. This phenyl group is highly flexibily, however, as only weak electron density was observed during crystal structure analysis<ref name=“main”/>. Such flexibility could be essential for substrates to cyclize. Also stabilizing the inhibitor is an arene-H interaction made between the imidazole moiety of PBD150 and W296. The first carbon upstream of this imidazole ring forms an additional arene-H contact with W176. Finally, the sulfur contained in the thiourea group makes a hydrogen bond with D271. This sulfur could mimic a carbonyl oxygen in the backbone of a peptide, suggesting a possible substrate binding mechanism.

===Catalytic Mechanism===

DromeQC is the enzyme responsible for this post-translational processing of polypeptides. DromeQC catalyzes the cyclization of N-terminal glutamine, and to a lesser extent glutamate, into pyroglutamic acid (5-oxo-L-proline, or <Glu) (Figure 5). This enzyme can be categorized as follows:

The cyclization reaction occurs via a nucleophilic attack of the α-amine on the γ carbon in the glutamine side chain. The enzymatic mechanism for DromeQC is still undetermined, but it seems plausible that it follows that of its human orthologue. In hQC, the N-terminus of the peptide substrate is inserted into the active site pocket, where the γ amide oxygen chelates the catalytic zinc ion<ref name="mechanism">PMID: 18470930</ref>. This ion-dipole interaction causes carbonyl polarization, making it a better electrophile. To facilitate the reaction, a conserved glutamate (Glu201) acts as both a general base and acid. Glu201 abstracts a proton from the α-amino group, causing it to nucleophilically attack the γ amide oxygen. This produces a tetrahedral intermediate with a charged oxygen that is stabilized by Zn²⁺. Glu201 then protonates the γ amide nitrogen, and an amine group is expelled as the carbonyl reforms. Also essential to this mechanism is a conserved aspartate (Asp248) that coordinates/stabilizes the leaving amine group.   

DromeQC is known to localize in the brain and peripheral nerves of ''D. melanogaster''<ref name="schilling"/>. This fact was unveiled by the discovery of adipokinetic hormone; this protein has an N-terminal pGlu, supporting a post translational modification via DromeQC<ref>PMID: 2117437</ref>. Adipokinetic hormone was found to localize in neurone and nerve endings by immunohistochemistry, suggesting that it functions as a locally released modulator in tissues such as heart and skeletal muscle<ref>PMID: 6342796</ref>. Thus, It has been suggested that DromeQC is part of the secretory pathway, being targeted to secretory vesicles where hormone maturation takes place<ref name="schilling"/>. This claim is supported by a 27 residue signal sequence contained at the N-terminus of DromeQC<ref name="schilling"/>. Further support stems from immunohistochemical evidence that DromeQC and its modified substrate are excreted from the cell, where they can be found in the extracellular medium<ref name="schilling"/>.